Brij B. Singh

TRPC1 Is Required for Functional Store-operated Ca2+ Channels ROLE OF ACIDIC AMINO ACID RESIDUES IN THE S5-S6 REGION

The exact role of TRPC1 in store-operated calcium influx channel (SOCC) function is not known. We have examined the effect of overexpression of full-length TRPC1, depletion of endogenous TRPC1, and expression of TRPC1 in which the proposed pore region (S5-S6, amino acids (aa) 557–620) was deleted or modified by site-directed mutagenesis on thapsigargin- and carbachol-stimulated SOCC activity in HSG cells. TRPC1 overexpression induced channel activity that was indistinguishable from the endogenous SOCC activity. Transfection with antisense hTRPC1 decreased SOCC activity although characteristics of SOCC-mediated current, ISOC, were not altered. Expression of TRPC1Δ567–793, but not TRPC1Δ664–793, induced a similar decrease in SOCC activity. Furthermore, TRPC1Δ567–793 was co-immunoprecipitated with endogenous TRPC1. Simultaneous substitutions of seven acidic aa in the S5-S6 region (Asp → Asn and Glu → Gln) decreased SOCC-mediated Ca2+, but not Na+, current and induced a left shift in E rev. Similar effects were induced by E576K or D581K, but not D581N or E615K, substitution. Furthermore, expressed TRPC1 proteins interacted with each other. Together, these data demonstrate that TRPC1 is required for generation of functional SOCC in HSG cells. We suggest that TRPC1 monomers co-assemble to form SOCC and that specific acidic aa residues in the proposed pore region of TRPC1 contribute to Ca2+ influx.

Calmodulin Regulates Ca2+-Dependent Feedback Inhibition of Store-Operated Ca2+ Influx by Interaction with a Site in the C Terminus of TrpC1

The mechanism involved in [Ca(2+)](i)-dependent feedback inhibition of store-operated Ca(2+) entry (SOCE) is not yet known. Expression of Ca(2+)-insensitive calmodulin (Mut-CaM) but not wild-type CaM increased SOCE and decreased its Ca(2+)-dependent inactivation. Expression of TrpC1 lacking C terminus aa 664-793 (TrpC1DeltaC) also attenuated Ca(2+)-dependent inactivation of SOCE. CaM interacted with endogenous and expressed TrpC1 and with GST-TrpC1 C terminus but not with TrpC1DeltaC. Two CaM binding domains, aa 715-749 and aa 758-793, were identified. Expression of TrpC1Delta758-793 but not TrpC1Delta715-749 mimicked the effects of TrpC1DeltaC and Mut-CaM on SOCE. These data demonstrate that CaM mediates Ca(2+)-dependent feedback inhibition of SOCE via binding to a domain in the C terminus of TrpC1. These findings reveal an integral role for TrpC1 in the regulation of SOCE.

Cytoskeletal Reorganization Internalizes Multiple Transient Receptor Potential Channels and Blocks Calcium Entry into Human Neutrophils

Store-operated calcium entry (SOCE) is required for polymorphonuclear neutrophil (PMN) activation in response to G protein-coupled agonists. Some immunocytes express proteins homologous to the Drosophila transient receptor potential gene (trp) calcium channel. TRP proteins assemble into heterotetrameric ion channels and are known to support SOCE in overexpression systems, but the evidence that TRP proteins support SOCE and are functionally important in wild-type cells remains indirect. We therefore studied the expression and function of TRP proteins in primary human PMN. TRPC1, TRPC3, TRPC4, and TRPC6 were all expressed as mRNA as well as membrane proteins. Immunofluorescence microscopy demonstrated localization of TRPC1, TRPC3, and TRPC4 to the PMN cell membrane and their internalization after cytoskeletal reorganization by calyculin A (CalyA). Either TRPC internalization by CalyA or treatment with the inositol triphosphate receptor inhibitor 2-aminoethoxydiphenyl borane resulted in the loss of PMN SOCE. Cytochalasin D (CytoD) disrupts actin filaments, thus preventing cytoskeletal reorganization, and pretreatment with CytoD rescued PMN SOCE from inhibition by CalyA. Comparative studies of CytoD and 2-aminoethoxydiphenyl borane inhibition of PMN cationic entry after thapsigargin or platelet-activating factor suggested that SOCE occurs through both calcium-specific and nonspecific pathways. Taken together, these studies suggest that the multiple TRPC proteins expressed by human PMN participate in the formation of at least two store-operated calcium channels that have differing ionic permeabilities and regulatory characteristics.

Trp1-dependent enhancement of salivary gland fluid secretion: role of store-operated calcium entry

This study examined the involvement of store‐operated Ca2+ entry in agonist‐stimulation of salivary gland fluid secretion. A recombinant adenovirus (AdCMV‐hTrp1) encoding the store‐operated Ca2+ channel protein, human transient receptor potential 1 (hTrp1), was used to direct expression of HA (hemaglutinin )‐tagged hTrp1 in vivo in rat submandibular glands (SMG) and in vitro in the human submandibular gland cell line (HSG). Studies with HSG cells demonstrated that AdCMV‐hTrp1 was successful in directing the expression of functional hTrp1 and that it did not affect early Ca2+ signaling events. AdCMV‐hTrp1‐infected SMG displayed an increase in the level of Trp1 and a fivefold increase in pilocarpine‐stimulated fluid secretion, compared with glands infected with a control adenovirus encoding luciferase (AdCMV‐Luc). The expressed hTrp1 demonstrated polarized localization in the basolateral plasma membrane region of SMG acinar cells and was co‐immunoprecipitated with IP3Rs. Further, acinar cells isolated from AdCMV‐hTrp1‐infected glands demonstrated a significant increase in carbachol‐ and Tg‐stimulated Ca2+ entry compared with cells isolated from AdCMV‐Luc‐infected glands. We conclude that in vivo expression of Trp1 in SMG induces an enhancement of agonist‐stimulated fluid secretion via increasing store‐operated Ca2+ entry into acinar cells. These data suggest that store‐operated Ca2+ entry has a role in agonist‐stimulated fluid secretion from salivary glands.

Expression of Truncated Transient Receptor Potential protein 1α (Trp1α) EVIDENCE THAT THE Trp1 C TERMINUS MODULATES STORE-OPERATED Ca2+ ENTRY

Transient receptor potential protein 1 (Trp1) has been proposed as a component of the store-operated Ca2+ entry (SOCE) channel. However, the exact mechanism by which Trp1 is regulated by store depletion is not known. Here, we examined the role of the Trp1 C-terminal domain in SOCE by expressing hTrp1α lacking amino acids 664–793 (ΔTrp1α) or full-length hTrp1α in the HSG (human submandibular gland) cell line. Both carbachol (CCh) and thapsigargin (Tg) activated sustained Ca2+ influx in control (nontransfected), ΔTrp1α-, and Trp1α-expressing cells. Sustained [Ca2+] i , following stimulation with either Tg or CCh in ΔTrp1α-expressing cells, was about 1.5–2-fold higher than in Trp1α-expressing cells and 4-fold higher than in control cells. Importantly, (i) basal Ca2+ influx and (ii) Tg- or CCh-stimulated internal Ca2+ release were similar in all the cells. A similar increase in Tg-stimulated Ca2+ influx was seen in cells expressing Δ2Trp1α, lacking the C-terminal domain amino acid 649–793, which includes the EWKFAR sequence. Further, both inositol 1,4,5-trisphosphate receptor-3 and caveolin-1 were immunoprecipitated with ΔTrp1α and Trp1α. In aggregate, these data suggest that (i) the EWKFAR sequence does not contribute significantly to the Trp1-associated increase in SOCE, and (ii) the Trp1 C-terminal region, amino acids 664–793, is involved in the modulation of SOCE.

ATP-DEPENDENT ACTIVATION OF KCA AND ROMK-TYPE KATP CHANNELS IN HUMAN SUBMANDIBULAR GLAND DUCTAL CELLS

[Ca2+] i and membrane current were measured in human submandibular gland ductal (HSG) cells to determine the regulation of salivary cell function by ATP. 1–10 μm ATP activated internal Ca2+ release, outward Ca2+-dependent K+ channel (KCa), and inward store-operated Ca2+ current (I SOC). The subsequent addition of 100 μm ATP activated an inwardly rectifying K+current, without increasing [Ca2+] i . The K+ current was also stimulated by ATP in cells treated with thapsigargin in a Ca2+-free medium and was blocked by glibenclamide and tolbutamide, but not by charybdotoxin. This suggests the involvement of a Ca2+-independent, sulfonylurea-sensitive K+ channel (KATP). UTP mimicked the low [ATP] effects, while benzoyl-ATP activated internal Ca2+ release, a Ca2+ influx pathway, and KCa. Thus, ATP acts via P2U (P2Y2) and P2Z (P2X7) receptors to increase [Ca2+] i and activate KCa, but not KATP. Importantly, (i) ROMK1 and the cystic fibrosis transmembrane regulator protein (but not SUR1, SUR2A, or SUR2B) and (ii) cAMP-stimulated Cl− and K+ currents were detected in HSG cells. These data demonstrate for the first time that a ROMK-type KATP channel is present in salivary gland duct cells that is regulated by extracellular ATP and possibly by the cystic fibrosis transmembrane regulator. This reveals a potentially novel mechanism for K+ secretion in these cells.

Valine 77 of heterocystous ferredoxin FdxH2 in Anabaena variabilis strain ATCC 29413 is critical for its oxygen sensitivity

Ferredoxins are small iron sulfur proteins necessary for electron donation. FdxH1 and FdxH2 are associated with two different nif gene clusters where they transfer electrons for the reduction of nitrogenase complex. FdxH1 was observed to be stable towards oxygen, whereas, FdxH2 was relatively unstable. We had identified the amino acid involved in oxygen sensitivity of ferredoxin protein using protein modeling. The exchange of valine to leucine at position 77 was critical for ferredoxin proteins in relation to its oxygen sensitivity. This exchange leads to a longer side chain, which inhibits the accessibility of oxygen to the iron sulfur cluster. Site directed mutagenesis and in vitro experiments confirms that valine indeed is involved in the oxygen sensitivity. The exchange of leucine to valine in FdxH1 makes it oxygen unstable. Thus, from the above results we can conclude that the position of leucine at position 77 is critical for oxygen sensitivity of ferredoxin and protein modeling can be used to identify specific amino acids in other oxygen-sensitive proteins.